This invention relates to nucleic acid molecules and amino acid sequences of a new mammalian protein and to their use in the characterization, diagnosis, prevention, and treatment of conditions such as hypertension, cancer, immune, and reproductive disorders.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical and structural features and modulate the same general activity. Comparisons of human gene sequences with those from other organisms where structure and/or function are known allow researchers to draw analogies and to develop model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Hypertension is a major cause of morbidity and mortality. It is probably the most important public health problem in developed countries, but the etiology is still largely unknown. As a result, treatment for hypertension may be nonspecific and lead to a large number of side effects and up to a 50 percent noncompliance rate. The prevalence of hypertension in the general population may vary by ethnicity, socioeconomic status, and gender. Dietary intake and genetic factors are also associated with the incidence rate of hypertension.
Hypertension is a common cause of chronic heart failure, particularly in older people whose heart muscle is weakened by age and progressive coronary valvular sclerosis. Fluid is retained by the kidneys to increase blood volume in compensation for the diminished pumping ability of the heart. Patients who develop malignant hypertension usually develop both heart and kidney failure.
Treatment of hypertension includes reduced sodium intake, weight loss, changes in living conditions, and treatment with drugs such as angiotensin II receptor antagonists, angiotensin converting enzyme inhibitors, diuretics, vasodilators, calcium channel antagonists, and antiadrenergic agents. Antiadrenergic agents may be classified into at least two groups, those which act upon the peripheral nervous system and those which act upon the central nervous system. The central acting agents are thought to act upon both adrenoreceptors and non-adrenoreceptors. Drugs such as clonidine bind to both the a2 adrenoreceptor and to a non-adrenoreceptor, the imidazoline receptor. The endogenous ligands for imidazoline receptors have been identified as agmatine, a decarboxylated form of the amino acid arginine (Herman, Z. S. (1997) Pol. J. Pharmacol. 49:85-88).
The discovery of mammalian nucleic acid molecules encoding an imidazoline receptor provides new compositions which are useful in the characterization, diagnosis, prevention, and treatment of hypertension, cancer, immune, and reproductive disorders.
The invention is based on the discovery of a substantially purified mammalian nucleic acid molecules encoding a mammalian imidazoline receptor (mIR), which satisfies a need in the art by providing compositions useful in the characterization, diagnosis, prevention, and treatment of conditions such as hypertension, cancer, immune, and reproductive disorders.
The invention provides isolated and purified mammalian nucleic acid molecules comprising the nucleotides 1 to 1424 and 2311 to 5128 of SEQ ID NO:1 or fragments thereof (SEQ ID NOs:3-12 and 18-29). The invention further provides fragments homologous to the mammalian nucleic acid molecule, SEQ ID NOs:30-46, in the Sequence Listing.
The invention further provides a probe which hybridizes under high stringency conditions to the mammalian nucleic acid molecule or fragments thereof. The invention also provides isolated and purified nucleic acid molecules which are complementary to the mammalian nucleic acid molecule comprising the nucleotides 1 to 1424 and2311 to 5128 of SEQ ID NO:1 or a fragment thereof (SEQ ID NOs:3-12 and 18-29) or a homologous nucleic acid (SEQ ID NOs:30-46). In one aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention further provides a method for detecting a nucleic acid molecule in a sample, the method comprising the steps of hybridizing a probe to at least one nucleic acid molecule of a sample, forming a hybridization complex; and detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of the nucleic acid molecule in the sample. In one aspect, the method further comprises amplifying the nucleic acid molecule prior to hybridization. The nucleic acid molecule or a fragment thereof may comprise either an element or a target on a microarray.
The invention also provides a method for using a nucleic acid molecule or a fragment thereof to screen a library of molecules to identify at least one ligand which specifically binds the nucleic acid molecule, the method comprising combining the nucleic acid molecule with a library of molecules under conditions allowing specific binding, and detecting specific binding, thereby identifying a ligand which specifically binds the nucleic acid molecule. Such libraries include DNA and RNA molecules, peptides, PNAs, proteins, and the like. In an analogous method, the nucleic acid molecule or a fragment thereof is used to purify a ligand.
The invention also provides an expression vector containing at least a fragment of the nucleic acid molecule. In another aspect, the expression vector is contained within a host cell. The invention further provides a method for producing a protein, the method comprising the steps of culturing the host cell under conditions for the expression of the protein and recovering the protein from the host cell culture.
The invention also provides a substantially purified mammalian imidazoline receptor or a portion thereof. The invention further provides isolated and purified proteins having the amino acid sequences of SEQ ID NO:2. Additionally, the invention provides a pharmaceutical composition comprising a substantially purified mammalian protein or a portion thereof in conjunction with a pharmaceutical carrier.
The invention further provides a method for using at least a portion of the mammalian protein to produce antibodies. The invention also provides a method for using a mammalian protein or a portion thereof to screen a library of molecules to identify at least one ligand which specifically binds the protein, the method comprising combining the protein with the library of molecules under conditions allowing specific binding, and detecting specific binding, thereby identifying a ligand which specifically binds the protein. Such libraries include DNA and RNA molecules, peptides, agonists, antagonists, antibodies, immunoglobulins, drug compounds, pharmaceutical agents, and other ligands. In one aspect, the ligand identified using the method modulates the activity of the mammalian protein. In an analogous method, the protein or a portion thereof is used to purify a ligand. The method involves combining the mammalian protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and separating the protein from the ligand to obtain purified ligand.
The invention further provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the natural mammalian nucleic acid molecule. The invention also provides a method for using the mammalian nucleic acid molecule to produce a mammalian model system, the method comprising constructing a vector containing the mammalian nucleic acid molecule; introducing the vector into a totipotent mammalian embryonic stem cell; selecting an embryonic stem cell with the vector integrated into genomic DNA; microinjecting the selected cell into a mammalian blastocyst, thereby forming a chimeric blastocyst; transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric mammal containing at least one additional copy of mammalian nucleic acid molecule in its germ line; and breeding the chimeric mammal to generate a homozygous mammalian model system.